The present invention relates to a fluorescent microscope which is mainly used in the medicine and biology, and in which the fluorescent dye of one specimen dyed with a lot of kinds of different fluorescent dyes is separated and an image of each fluorescent dye is separately imaged with an imaging element of black and white, thereafter the image of each fluorescent dye is overlapped and observed at the same time.
In general, a reflected light fluorescent microscope widely used to detect the protein and the gene, etc. to which a fluorescent marker is performed on the bio-organ and the cell in several fields including medicine and biology.
Especially, in recent years, by developing various fluorescent dyes, the reflected light fluorescent microscope is used to examine position interrelationship of specific material and localization of specific material in cell by a multiple dye to mark and identify various materials with a different fluorescent dye.
By the way, conventionally, when the multiple dye specimen is observed with this reflected light fluorescent microscope, the fluorescent filter set in which an excitation filter, a dichroic mirror, and the barrier filter to observe a single fluorescent dye are to be one set, is prepared for each specific fluorescent dye, beforehand, respectively, and specific fluorescent dyes are observed one by one by changing the fluorescent filter set to be the best combination for the fluorescent dye. And, when the position of each fluorescent dye is checked, each fluorescent image is multiple recorded and displayed in a photograph and a video memory.
However, in the above-mentioned reflected light fluorescent microscope, not only it becomes expensive but also it takes time to operate the change of a fluorescent filter, since it is necessary to prepare many kinds of fluorescent filter sets. A large space is required for the device and the operation.
On the other hand, there is a method of observing the distribution of the density of the ion in the cell and the target change at the time of passing as an image by using the fluorescent dyes which change the excitation spectrum (wavelength excited easily) reacting on the change of the density of the ion such as Ca.sup.2+ ions disclosed in Japanese Patent Application KOKAI Publication No. 2-28542 (whose corresponding U.S. patent application, which has been withdrawn, is Ser. No. 08/340,236, filed Apr. 19, 1989). In this method, the distribution of the density of the ion is displayed by preparing the two kinds of excitation filters which the wavelength to which fluorescent dyes are excited easily most at each state of certain different two specific ion densities, changing the excitation filters, and calculating the ratio of fluorescent strength in each state of excitation.
Since the change in the characteristic of the fluorescent dye can be obtained by changing only the excitation filter without changing the fluorescent filter set, the little number of pieces of the filters are required and cheapness and occupied space can be reduced according to this method.
However, this method is a method of identifying the change in the characteristic of one kind of a fluorescent dye, but is not a method of identifying different kinds of fluorescent dyes. It is necessary to prepare the same number of the excitation filters as the kinds of the fluorescent dyes which try to be identified and perform the change operation even when trying to apply to the identification of the kind of the fluorescent dye. Therefore, a disadvantage of becoming expensive with requiring many filters entirely and a disadvantage of taking time for the change operation, and needing a large space cannot be solved as a result.
In addition, means to change the transmitting wavelength band of the filter without changing the filter, in which this technique is well-known in the prior art, has been described in the U.S. Pat. No. 5,371,624. In both of these publications, the transmitting wavelength band is changed by combining with the fluorescent filter set with the plurality of transmitting wavelength band to observe a plurality of fluorescent dyes at the same time, in addition, the excitation filter or the barrier filter which uses another interference film is prepared, and changing the length of the interference optical path by rotating the excitation filter or the barrier filter around a rotation axis vertical to the optical axis.
However, these methods are the methods to adjust the balance of the brightness of the fluorescence of each fluorescent dye suitably, and to make easily to observe when a plurality of fluorescent dyes are observed at the same time, but not the methods of separating a different fluorescent dye and identifying it.
The fluorescent filter set, in which a plurality of different fluorescent dyes can be observed at the same time without changing the fluorescent filter set is described in the publications as mentioned above. There are a method of imaging a fluorescent image with the imaging element of the color and a method of imaging a fluorescent image with the imaging element of black and white by using the Michelson interferometer and the diffraction grating when the fluorescent image is imaged.
However, a disadvantage in which sensitivity is insufficient and the exposure time not only becomes a long in the color imaging element but also the noise component in the image increases, and the resolution is deteriorated since a fluorescent image is generally slight light is raised.
Since the spectrum device becomes large-scale and expensive in the method of imaging a fluorescent image with the monochrome imaging element, not only a large space for the installation but also a long time are required to obtain the spectrum. Therefore it is still undesirable.
In addition, each of both methods of imaging with the above-mentioned color imaging element and imaging with the imaging element of black and white is a method of comparing the spectrum intensity characteristic of a fluorescent image with the spectrum characteristics of the luminescence fluorescence of the fluorescent dye to specify the fluorescent dye, and there is a disadvantage of missing to accuracy.